The bacterial phosphoenolpyruvateglycose phosphotransferase system (PTS) comprises a group of proteins that catalyze the transfer of the phosphoryl group from PEP to sugars, concomitant with their translocation. The first two steps of the phosphotransfer sequence are: PEP ~ Enzyme I ~ HPr. Many functions of the PTS are regulated by EI, which undergoes a monomer/dimer transition. EI monomer, (63.5 kDal) comprises two major domains: a flexible C-terminal domain (EI-C), and a protease-resistant, structurally stable N-terminal domain (EI-N), containing the active site His. Trypsin treatment of Salmonella typhimurium EI yields EI-N, designated EI-N(t). Homogeneous recombinant Escherichia coli , designated EI-N(r), has been now prepared in quantity and shows the expected thermodynamic unfolding properties, and, similarly to EI-N(t), is phosphorylated by phospho-HPr, but not by PEP. In addition, binding of EI-N(r) to HPr was studied by isothermal titration calorimetry: K~a= 1.4 x 105 M-1 and DH = +8.8 kcal.mol-1. Both values are comparable to those for HPr binding to intact EI. Fluorescence anisotropy and gel filtration of EI-N(r) show that it does not dimerize. These results emphasize the role of EI-C in dimerization and the regulation of intact EI.